Cancer is defined by dysregulated signaling, enabling cells to sustain proliferation. Phosphotyrosine signaling plays a central role in normal and aberrant signaling networks with a majority of tyrosine kinases implicated in cancer. Differential phosphorylation of the proto-oncogene Src has been observed in numerous cancers including lung, breast, and colon, but technological challenges have limited study of specific phosphorylated residues. Analysis of phosphorylation on tyrosine, serine, and threonine residues has been limited by our inability to control these chemical modifications in vivo or in vitro due to a lack of phosphomimetics that fully recapitulate the chemistry of phoshorylated residues. Recent work has demonstrated direct co-translational incorporation of phosphoserine into mitogen-activated protein kinase kinase (Mek), enabling production of recombinant phosphorylated MEK without co-expression of regulatory proteins. In this scheme, non-standard amino acids like phosphoserine are incorporated via a paired aminoacyl-tRNA synthetase and tRNA directed to a TAG stop codon. No analogous system exists for phosphotyrosine. We have furthered this work through the generation of a genomically recoded E. coli, that is optimized for the incorporation of phosphoserine. We hypothesize that aminoacyl-tRNA synthetase evolution in the recoded E. coli will enable site-specific incorporation of phosphotyrosine. We will apply this technological advance to deciphering regulation of Src, where previous assays have been limited by inhomogeneously phosphorylated protein. We hypothesize that Src phosphorylated exclusively on its C-terminus domain (Tyr527) will show complete inactivation, while the acquisition of a second phosphorylation event on its SH2 domain (Tyr213) will lead to recovery of function. Specific Aims: This proposal includes two specific aims. The first is to utilize mutagenesis to engineer an optimized tyrosyl aminoacyl-tRNA synthetase for amino acids structurally similar to phosphotyrosine. We will then use metabolic engineering to produce a stable phosphotyrosine pool in the recoded E. coli. Finally, we will integrate these components to generate phosphotyrosine-containing proteins, confirming incorporation by mass spectrometry. In the second aim, we will apply this co-translational phosphotyrosine incorporation system to the biochemical characterization of Src. We will produce Src phosphorylated uniquely on its C-terminal inhibitory domain and characterize kinase activity against a series of known substrates in vitro. To study the effects of SH2 domain phosphorylation on inhibited Src, we will express Src dually phosphorylated on the SH2 and inhibitory domains, hypothesizing that SH2 phosphorylation can overcome intramolecular Src inhibition. Relevance: The overall aim of this proposal is to develop and apply foundational technologies to decoding phosphotyrosine-mediated regulation of the proto-oncogene tyrosine kinase Src. We seek to assess the individual contributions of phosphorylation events on Src to reveal physiologically relevant mechanisms of activation.